The present invention relates generally to the field of lipid biochemistry. More specifically, the present invention relates to the cloning of human choline/ethanolaminephosphotransferases.
Cholinephosphotransferase catalyzes the final step in the synthesis of PtdCho through the Kennedy (CDP-choline) pathway via the transfer of a phosphocholine moiety from CDP-choline to diacylglycerol with the release of CMP and the formation of PtdCho (Vance, 1996 in Biochemistry of Lipids, Lipoproteins and Membranes, Vance, D E and Vance, J E, Elsevier: Amsterdam, pp 153-182; Kennedy and Weiss, 1956, J Biol Chem 222:193-214; Weiss et al, 1958, J Biol Chem 231:53-64; McMaster and Bell, 1997, Biochim Biophys Acta 1348:100-110; Hjelmstad and Bell, 1991, J Biol Chem 266:4357-4365; McMaster and Bell, 1994, J Biol Chem 269:28010-28016; and Cornell, 1989 in Phosphatidylcholine Metabolism, Vance, D E, Boca Raton: Fla., pp. 47-64). The fatty acyl composition of the diacylglycerol molecule utilized by cholinephosphotransferase determines the fatty acyl array for de novo synthesized PtdCho. In addition, the intracellular location of cholinephosphotransferase identifies the site of de novo PtdCho synthesis for subsequent transfer to other organelles, or assembly with proteins and other lipids for secretion during the synthesis of lung surfactant, lipoproteins, and bile (Jobe, 1993, N Engl J Med 328:861-868; Steinberg, 1997, J Biol Chem 272 2Q963-20966; Ruetz and Gros, 1994, Cell 77:1071-1081).
Properties of cholinephosphotransferase activities present in membrane preparations have identified an activity capable of de novo synthesis of the PtdCho structural analogues, platelet activating factor (PAF), a major mediator of inflammatory processes, as well as PAF precursor (Snyder, 1997, Biochim Biophys Acta 1348:111-116; Leslie, 1997, J Biol Chem 272:16709-16712; Venable et al, 1993, J Lipid Res 34:691-702). However, the biological and biochemical roles of a de novo synthesized PAF pathway, and indeed the roles of the various proposed isoforms of cholinephosphotransferase in the regulation of the partitioning of lipid biosynthetic pathways, have yet to be determined as a mammalian cholinephosphotransferase has not been cloned or purified.
Genetic approaches led to the isolation of two genes encoding cholinephosphotransferase activities from the yeast Saccharomyces cerevisiae. The yeast CPT1 gene product encodes a cholinephosphotransferase (McMaster and Bell, 1994; Hjelmstad and Beg, 1990, J Biol Chem 265:1755-1764: Hjelmstad and Bell, 1987, J Biol Chem 262:3909-3917) specific for the synthesis of PtdCho in vitro and in vivo, while the EPT1 gene product codes for a dual specificity choline/ethanolaminephosphotransferase capable of synthesizing PtdCho and phosphatidylethanolamine (PtdEtn) in vitro, but which synthesizes primarily PtdEtn in vivo (McMaster and Bell, 1994; Hjelmstad and Bell, 1991, J Biol Chem 266:5094-5103). Analysis of chimeric CPT1/EPT1 enzymes (McMaster and Bell, 1994; Hjelmstad and Bell, 1991; Hjelmstad and Bell, 1988. J Biol Chem 263:19748-19757) mapped the active site domain, and site-directed mutagenesis identified a diagnostic catalytic motif (Hjelmstad et al, 1994, J Biol Chem 269:2009521002). It was hypothesized that active site residues would be conserved between Genera. This rationale was used as a basis to isolate human choline/ethanolaminephosphotransferase cDNAs (hCEPT1 and hCEPT2) for subsequent expression and characterization.
According to a first aspect of the invention, there is provided an isolated native, cloned, recombinant or synthetic DNA sequence encoding hCEPT1 protein comprising nucleotides 497-2051 of SEQ ID NO,: or sub-fragments thereof.
According to a second aspect of the invention, there is provided an hCEPT1 protein having an amino acid sequence substantially as shown in SEQ ID NO:2.
According to a third aspect of the invention, there is provided a DNA molecule encoding hCEPT1 protein, said DNA deduced from the amino acid sequence of SEQ ID NO2.
According to a fourth aspect of the invention, there is provided an isolated native, cloned, recombinant or synthetic DNA sequence encoding hCEPT2 protein comprising nucleotides 1-881 of SEQ ID NO:3 or sub-fragments thereof.
According to a fifth aspect of the invention, there is provided hCEPT2 protein having an amino acid sequence substantially as shown in SEQ ID NO:4.
According to a sixth aspect of the invention, there is provided a DNA molecule encoding hCEPT1 protein, said DNA deduced from the amino acid sequence according to SEQ ID NO:4.
According to a seventh aspect of the invention, there is provided a recombinant expression system, capable, when transformed into a host cell, of expressing a DNA sequence encoding hCEPT1 (SEQ ID NO:2) or hCEPT2 (SEQ ID NO:3) which system comprises control sequences effective in said host cell operably linked to said DNA sequence.
According to an eighth aspect of the invention, there is provided a host cell transformed with the expression system described above.
The host cell may be selected from the group consisting of: a plant cell; a yeast cell; a bacterial cell; and a mammalian cell.
According to a ninth aspect of the invention, there is provided mutant hCEPT1 protein having an amino acid sequence substantially as shown in FIG. 2A (SEQ ID NO:2) and having a missense mutation at glycine 156.
According to a tenth aspect of the invention, there is provided a DNA molecule encoding mutant hCEPT1 protein, said DNA deduced from the amino acid sequence described above.
According to an eleventh aspect of the invention, there is provided mutant hCEPT2 protein having an amino acid sequence substantially as shown in FIG. 2B (SEQ ID NO:4) and having a missense mutation at glycine 156.
According to a twelfth aspect of the invention, there is provided a DNA molecule encoding mutant hCEPT2 protein, said DNA deduced from the amino acid sequence described above.
According to a thirteenth aspect of the invention, there are provided antibodies directed against hCEPT, said hCEPT selected from the group consisting of: hCEPT1 (SEQ ID NO:2); hCEPT2 (SEQ ID NO:4); mutant hCEPT1 protein having an amino acid sequence substantially as shown in SEQ ID NO:2 and having a missense mutation at glycine 156; mutant hCEPT2 protein having an amino acid sequence substantially as shown in SEQ ID NO:4 and having a missense mutation at glycine 156; and immunoreactive fragments thereof.
The antibodies described above may be used to identify proteins related to hCEPT1 and hCEPT2 or for diagnostic use.
According to a fourteenth aspect of the invention, there is provided a method of synthesizing lipids containing a given fatty acid composition comprising:
providing hCEPT protein, selected from the group consisting of: hCEPT1 (SEQ ID NO:2); hCEPT2 (SEQ ID NO:4); mutant hCEPT1 protein having an amino acid sequence substantially as shown in SEQ ID NO:2 and having a missense mutation at glycine 156; mutant hCEPT2 protein having an amino acid sequence substantially as shown in SEQ ID NO:4 and having a missense mutation at glycine 156; and combinations thereof;
providing substrates required for lipid biosynthesis;
combining the hCEPT protein and the substrates;
incubating the hCEPT protein and the substrates under conditions promoting lipid biosynthesis; and
harvesting the lipids.
According to a fifteenth aspect of the invention, there is provided lipids prepared according to the above-described method.
The lipids may be used as a food additive.
According to a sixteenth aspect of the invention, there is provided a method of assaying a compound for modulation of lipid metabolism comprising:
providing hCEPT protein, selected from the group consisting of: hCEPT1 (SEQ ID NO:2); hCEPT2 (SEQ ID NO:4); mutant hCEPT1 protein having an amino acid sequence substantially as shown in SEQ ID NO:2 and having a missense mutation at glycine 156; mutant hCEPT2 protein having an amino acid sequence substantially as shown in SEQ ID NO:4 and having a missense mutation at glycine 156; and combinations thereof;
providing substrates required for lipid biosynthesis;
providing a compound proposed to modulate lipid metabolism;
combining the compound, the hCEPT protein and the substrates;
incubating the compound, the hCEPT protein and the substrates under conditions promoting lipid biosynthesis.
harvesting the lipids;
and characterizing the lipids, thereby determining the effect of the compound on lipid metabolism.
According to a seventeenth aspect of the invention, there is provided a reagent for use in disease diagnosis or genotyping comprising antibodies directed against hCEPT, said hCEPT selected from the group consisting of: hCEPT1 (SEQ ID NO:2); hCEPT2 (SEQ ID NO:4); mutant hCEPT1 protein having an amino acid sequence substantially as shown in SEQ ID NO:2 and having a missense mutation at glycine 156; mutant hCEPT2 protein having an amino acid sequence substantially as shown in SEQ ID NO:4 and having a missense mutation at glycine 156; and immunoreactive fragments thereof.
According to an eighteenth aspect of the invention, there is provided a reagent for use in identifying proteins related to hCEPT comprising antibodies directed against hCEPT, said hCEPT selected from the group consisting of: hCEPT1 (SEQ ID NO:2); hCEPT2 (SEQ ID NO: 4); mutant hCEPT1 protein having an amino acid sequence substantially as shown in SEQ ID NO:2 and having a missense mutation at glycine 156; mutant hCEPT2 protein having an amino acid sequence substantially as shown in SEQ ID NO:4 and having a missense mutation at glycine 156; and immunoreactive fragments thereof.
According to a nineteenth aspect of the invention, there is provided a nucleotide probe selected from the group consisting of: nucleotides 497-2051 of SEQ ID NO:1; nucleotides 1-881 of SEQ ID NO:3; and fragments thereof.
According to a twentieth aspect of the invention, there is provided an oligonucleotide for use in identifying genes related to hCEPT, said oligonucleotide selected from the group consisting of: nucleotides 497-2051 of SEQ ID NO:1; nucleotides 1-881 of SEQ ID NO:3; and fragments thereof.
According to a twenty-first aspect of the invention, there is provided an antisense probe for use in treating lipid metabolic disorders, said antisense probe being complementary to nucleotides 497-2051 of SEQ ID NO:1; nucleotides 1-881of SEQ ID NO:3; or fragments thereof.
One embodiment of the invention will now be described in conjunction with the accompanying figures in which: